Genetic analysis of chorionic villus tissues in early missed abortions

Chromosomal abnormalities are the most common etiology of early spontaneous miscarriage. However, traditional karyotyping of chorionic villus samples (CVSs) is limited by cell culture and its low resolution. The objective of our study was to investigate the efficiency of molecular karyotyping technology for genetic diagnosis of early missed abortion tissues. Chromosome analysis of 1191 abortion CVSs in early pregnancy was conducted from August 2016 to June 2021; 463 cases were conducted via copy-number variations sequencing (CNV-seq)/quantitative fluorescent-polymerase chain reaction (QF-PCR) and 728 cases were conducted using SNP array. Clinically significant CNVs of CVSs were identified to clarify the cause of miscarriage and to guide the couples’ subsequent pregnancies. Among these, 31 cases with significant maternal cell contamination were removed from the study. Among the remaining 1160 samples, 751 cases (64.7%) with genetic abnormalities were identified, of which, 531 (45.8%) were single aneuploidies, 31 (2.7%) were multiple aneuploidies, 50 (4.3%) were polyploidies, 54 (4.7%) were partial aneuploidies, 77 (6.6%) had submicroscopic CNVs (including 25 with clinically significant CNVs and 52 had variants of uncertain significance), and 8 cases (0.7%) were uniparental disomies. Our study suggests that both SNP array and CNV-seq/QF-PCR are reliable, robust, and high-resolution technologies for genetic diagnosis of miscarriage.


CNV sequencing/QF-PCR
According to its detection principle, CNV-seq alone cannot recognize SNP site to exclude MCC.Thus, for CNV-Seq technology, samples should be first investigated using QF-PCR assays or STR test to exclude significant MCC and triploidy.
CVSs samples were subjected for CNV-seq after exclusion of MCC.A total of 449 cases of chromosomal analysis was ultimately conducted by CNV sequencing (CNV-seq) due to 14 cases with significant MCC.Briefly, 10 mg of CVSs were selected under sterile conditions, DNA was extracted by Qiagen kit for later use, DNA library was constructed using dsDNA fragmentase to produce smaller fragments, end repair, A-tail and then ligated with barcoded sequencing adaptors.Single-end sequencing was performed on the the NextSeq CN500 platform to produce to produce approximately 5 million reads per sample.The number of reads after sample quality control is more than 8 M Sequence depth is ~ 1x.Each sequencing tag was matched to the corresponding chromosome by comparison software, and then standardized Z-value analysis was performed to determine chromosome copy number variation.On average, approximately 2.8 ~ 3.2 million reads were uniquely mapped for data analysis.The uniquely mapped reads were aligned to the human genomic reference sequences (hg19) using the Burrows-Wheeler algorithm and allocated to 20-kilobase (kb) bin on each chromosome 14 .To eliminate the influence of GC bias between different samples, GC correction was performed via LOESS regression, intrarun normalization, and linear model regression 15 , and CNVs were identified from 24 chromosome copy number plots.The pathogenicity of detected CNVs was assessed following American College of Medical Genetics (ACMG) guidelines.Genomic variant databases including DGV, DECIPHER , OMIM, PubMed and UCSC were used as a reference source of CNVs 16 .
The CNV-seq data analysis processes include (1) Quality control of raw sequencing data (fastq), removing adapters/low-quality bases, resulting in filtered fastq data.(2) Alignment of filtered fastq data to the human reference genome (hg19) using alignment software like BWA. (3) Sorting and deduplication of the alignment results.(4) Binning the sorted results using genomic intervals to obtain read-depth profiles (sample-wise median normalization is applied, where the reads count in each bin is divided by the median reads count of the bins in autosomes.This step aims to mitigate biases introduced by different sequencing depths.( 5) GC correction of the read-depth profiles.( 6) CNV calling based on the GC-corrected read-depth profiles to detect CNV variations.(7) Analysis and interpretation of CNVs according to ACMG/ClinGen guidelines, including classification based on their clinical significance.

SNP array
Due to the platform transition in our laboratory during the study period, totally, 711 cases of chromosomal CNVs (above 0.1 Mb) analysis were ultimately detected by SNP array due to 17 cases with significant MCC.The experimental process of SNP array was carried out using Affymetrix CytoScan 750 K array (Affymetrix Inc., Santa Clara, CA) as previously described 17 .The data was analyzed using Affymetrix Chromosome Analysis Suite Software (version 3.1.0.15).Parental microarray testing was carried out to determine its origin.Pathogenicity of CNVs is classified according to ACMG guideline 16 .Only P/LP CNVs and VOUS are reported in this study.Large CNVs (≥ 10 Mb) were classified as partial aneuploidies, and submicroscopic CNVs (< 10 Mb) were defined as microdeletions and microduplications.Large CNVs detected in three or more cases were defined as recurrent large CNVs, while submicroscopic CNVs found in two or more cases were considered as recurrent submicroscopic CNVs.

Parental conventional karyotyping analysis
Peripheral blood lymphocyte was isolated from the abortion couples when the chromosomal aberrations of CVSs suggesting parental balanced rearrangement, then were cultured and harvested after phytohemagglutinin stimulation for 72 h.Metaphase chromosomes were prepared following standard cytogenetic protocols.

Statistical analysis
SPSS software version 19.0 (SPSS, Inc., Chicago, IL) was used for statistical analysis.Measurement data were expressed as mean ± standard deviation, statistical comparisons were performed using χ 2 test (Fisher's exact test), and p < 0.05 was considered statistically significant.

Ethics approval and consent to participate
The study complied with the principles set forth in the Declaration of Helsinki.It was approved by the Institutional Review Board of Fujian Maternal and Child Health Hospital.Written informed consent was obtained from each patient.

Baseline characteristics of the participants and chromosomal abnormalities of CVSs detected by molecular karyotyping technologies
Initially, we analyzed a total of 1191 cases by CNV-seq/QF-PCR or SNP array August 2016 to June 2021.Among these, 31 cases with serious MCC were removed.The remaining 1160 cases with no MCC were available for further molecular karyotyping analysis (Table 1), of which 449 cases were tested via CNV-seq/QF-PCR, and 711 cases were tested using SNP array.All 1160 CVSs were successfully analyzed.Thus, the detection success rate was 100% (1160/1160).As detection coverage was theoretically consistent except uniparental disomy (UPD) based on SNP array and CNV-seq/QF-PCR, we counted the results except UPD detected by the two methods together.Overall, 751 cases (64.7%) with genetic abnormalities were identified, of which, 531 (45.8%) were single aneuploidies, 31 (2.7%) were multiple aneuploidies, 50 (4.3%)were polyploidies, 54 (4.7%) were partial aneuploidies, 77 (6.6%) had CNVs (including 52 (4.5%) had VOUS), and 8 cases (0.7%) were UPD (Fig. 1).In addition, a total of 213 female samples and 196 male samples were found in the normal samples, with a female-to-male ratio of 1.09.
Polyploidy was found in 50 (4.3%)cases: triploidy in 45 (3.9%) cases and hypotriploidy in 5 (0.4%) cases (Table 2).3).Both large deletions and duplications occurred most commonly in chromosome 8, terminal deletion/duplication was identified in 18 (1.6%)cases, accounting for the largest proportion of partial aneuploidies.The distributions of large CNVs and associated genes in CVSs are shown in Table 3.Large copy number losses occurred mostly in chromosome 8, followed by chromosomes X and 7. Large copy number gains also occurred most frequently in chromosome 8, followed by chromosome 9.In addition, a terminal deletion accompanied by terminal duplication involving two chromosomes was detected in 17 cases, suggesting the presence of an unbalanced translocation, which represented the second largest proportion of partial aneuploidies (17 cases; 31.5% of all partial aneuploidies and 1.5% of all analyzed cases), among these, the karyotypes of 16 couples were available, and 14 couples were reported as reciprocal translocation carriers, except for 2 that had normal karyotypes.Samples from the two couples with the normal karyotypes were further subjected to FISH analysis, and two (Case 31 and 54) of them were identified to have submicroscopic reciprocal balanced translocations (see Table 3 and Fig. 3).A terminal   www.nature.com/scientificreports/deletion coupled with terminal duplication involving one chromosome was detected in 8 cases, suggesting an unbalanced pericentric inversion, which accounted for 14.8% of all partial aneuploidies and 0.7% of all analyzed cases, further parental studies showed 5 of them were derived from inversion) (Table 3), in the remaining 23 cases, parental studies were not available due to their refusal.Submicroscopic CNVs (< 10 Mb) (microdeletions/ microduplications) were identified in 77 (6.6%) cases.The distributions of submicroscopic P/LP CNVs and associated genes in CVSs are shown in Table 4.Among these, 22 (1.9%) were considered pCNVs, and 3 (0.3%) and 52 (4.5%) were classified as likely pCNVs and VOUS, respectively (Table 2).
Thirty-nine and twenty-nine cases with pCNVs were found in ≤ 29-year-old and 30-34-year-old pregnant women, respectively.There were only 7 and 2 pCNVs in CVSs detected in the 35-39 and ≥ 40-year-old age groups (Fig. 4).The number of chromosomal variations in the subgroups with previous miscarriages is shown in Fig. 5.There were no significant differences in the different types of chromosomal abnormalities among the four groups (p > 0.05).

The associations between chromosomal abnormalities of CVSs and maternal age
We investigated the relationship between maternal age and chromosomal abnormalities, finding that the frequency of aneuploidy increased with maternal age.The incidence of chromosomal aneuploidy abnormalities among women aged ≤ 29, 30-34, and 35-39 years was all significantly lower than that in women ≥ 40 years old (p < 0.001), whereas the frequencies of other types of chromosomal abnormalities were not correlated with maternal age (p > 0.05).The correlation between chromosomal abnormalities and maternal age is presented in Fig. 4.

Discussion
Early pregnancy loss puts a heavy psychological burden on most couples.Embryonic numerical and structural chromosomal abnormalities are the most common cause of early pregnancy loss 6,19 .Chromosomal aberration analysis of CVSs is essential for assessing the causes of first-trimester miscarriages.In the current study, we aimed to evaluate the incidence and distribution of chromosomal abnormalities detected by CMA and CNVseq/QF-PCR in CVSs samples.
Until now, karyotyping, CMA, as well as CNV-seq were the main technologies for identifying chromosomal anomalies [19][20][21] .Karyotyping is considered the "gold standard" for cytogenetics, its advantage is that it can detect chromosomal numerical, and visible structural abnormalities.However, this technique requires cell culture.Furthermore, its resolution is too low to detect submicroscopic CNVs.Thus, the total detection failure rate is ~ 20% 22 , which sometimes yields false-negative results due to the excessive growth of maternal cells relative to chorionic villus cells.CMA is a first-tier method in detecting CNVs in spontaneous miscarriages 23 .Although SNP array has significant advantages in detecting non-equilibrium chromosomal abnormalities, triploidy, and UPD, it has a relatively low throughput and a high detection cost.Recently, CNV-seq is gradually being used in detecting the genetic causes of CVSs 24 .Both molecular karyotyping technologies do not require cell culture and are reliable in detecting CNVs 25 .Compared with CMA, CNV-seq/QF-PCR has a higher throughput, a more accurate CNV breakpoint detection with 1 × depth, low cost, and a short reporting period, and has a lower level (≥ 10%) of mosaicism and polyploidy detection.We summarized the total detection efficiencies of SNP array and CNV-seq technologies on CVSs.In this study, all 449 and 711 CVSs were successfully examined via CNV-seq/ QF-PCR and SNP array, respectively, thus, the detection success rate of both methods was 100%.More normal female samples were observed than normal male samples (213 vs. 196), which was likely caused by confusion of 69 XXX and 46 XX, which is significantly higher than the mean ratio of 0.71 for cases investigated in multiple large-scale products-of-conception studies using cytogenetic analysis 26 .
The overall rate of clinically significant chromosomal abnormalities was 59.7% (693/1160) (Table 2), which is similar to the reported rates in previous researches 27 .Chromosomal numerical anomaly is the most common genetic cause of spontaneous miscarriages 28 .Here, 615 cases (81.9%, 615/751) had numerical abnormalities, which is consistent with results from a recent report 9 , including 531 cases (45.8%) of single chromosome aneuploidy and 31 (2.7%) cases of multiple aneuploidies (Table 2).The occurrence frequency of each chromosome aneuploidy was analyzed in all 531 cases in this study (Fig. 2), which showed that non-segregation errors occurred in all chromosomes except chromosome 1, which is consistent with a previous study 29 .
As the longest human chromosome, trisomy 1 appears to be a very early lethal anomaly that may affect early embryonic development, causing pregnancy failure or biochemical pregnancy 30 .As shown in Fig. 2, trisomy 16 accounts for 81% (430/531) of autosomal trisomy cases, which is in line with the previous report 31 .Among the cases of single autosomal trisomy, the incidence of trisomy 16 was the highest (18.7%, 115/615), followed by trisomy 22 and trisomy 21 (Fig. 2), which is consistent with the reported research 6 .Non-separation of homologous chromosomes causes aneuploidies during the meiotic divisions of germ cells 32 .The development and survival of zygotes with trisomies are inhibited, resulting in early pregnancy loss.Triploidy is a common chromosomal aberration that usually results in early miscarriage 33 .In our study, polyploidy was found in 50 (4.3%)cases: triploidy in 45 (3.9%) cases, and hypotriploidy in 5 (0.4%) cases, which are slightly lower than those reported by Wang Y. 6 This difference may be due to inadequate sample size in our study.
Monosomies are lethal due to their gene dose effect; thus, embryonic monosomies often occur very early in pregnancy and lead to spontaneous abortions 34 .Here, monosomies were identified in 19.0% (101/531) of the cases with single aneuploidy, of which, monosomy X accounted for 91.1% (92/101).Unlike autosomal trisomy, the risk of monosomy X did not increase with maternal age, consistent with previous reports 3 .Hassold et al. 35 found that the paternal loss of a sex chromosome was the most common reason leading to 45, X, which was likely a result of meiotic errors.
The third-child policy in China led to an increasing proportion of AMA.The risk of chromosomal aneuploidies in aborted embryos increases with maternal age 36 .This may be due to egg aging which can cause meiosis without dissociation 36 .Our data also support this point.
Generally, large CNVs which contain numerous genes are expected to be lethal and are known to cause miscarriage 6 .In this study, 79 large CNVs (≥ 10 Mb) in 54 cases were identified.Among 54 cases of large CNVs, thirty were unbalanced rearrangements, with 21 of them confirmed to be of parental origin (16 cases of translocations and 5 cases of inversion) in the subsequent conventional karyotyping/FISH of couples.In addition, we also found that both large deletions and duplications occurred most commonly in chromosome 8, which is consistent with the recent report 6,37 .One likely explanation is that maternal 8p inversion, which is delimited by the olfactory receptor (OR) gene clusters, and may confer susceptibility to unequal crossovers between two OR gene clusters 38 .The number of previous miscarriages ranged from 0 to 4. Approximately 2.7-6.7% of chromosomal balanced rearrangement carriers have experienced recurrent spontaneous abortion (RSA), which indicates that chromosomal balanced rearrangement is one of the main causes of RSA.Our data indicate that the genetic cause of miscarriage should be conducted routinely, abnormal CVSs results can suggest balanced abnormalities in parents, and follow-up parental testing allows for further refining recurrence risk and whether or not prenatal and/or pre-implantation genetic testing (PGT) needs to be conducted 39 .With exact chromosomal diagnosis, PGT was recommended to these couples 40 .We emphasized that an obstetrician should be aware of submicroscopic reciprocal translocation rearrangement in couples with RSA.
It is worth pointing out that in 2 couples with normal karyotypes, subsequent peripheral blood FISH was performed to confirm whether there is occult chromosomal rearrangement; both cases were proven to be hereditary from the parental submicroscopic reciprocal balanced translocations (Cases 30 and 53).Thus, submicroscopic  www.nature.com/scientificreports/reciprocal translocation, even in embryotic large pCNVs in couples with RSA, were sometimes ignored.Further FISH confirmation is recommended for carriers with suspected occult translocations or similar terminal chromosomal banding involving two chromosomes.However, the role of submicroscopic CNVs in early pregnancy loss is unclear.Specific information based on large cohorts regarding the association between submicroscopic CNVs and miscarriage is limited.Nevertheless, recent studies have shown that dosage changes or mutations of genes that play an important role in early embryonic development could also cause miscarriage 41 .A total of 77 (6.6%) cases of submicroscopic (< 10 Mb) CNVs were identified in the study, similar to those in Levy B's research 37 .Up to now, little is known about the association between a specific submicroscopic pCNV and miscarriage.
We identified submicroscopic pCNVs in 2.2% (25/1160) of our cases, which is same to 2.2% by Wang et al. 6 .Three recurrent pCNVs, including 22q11.2 microdeletion, 7q11.23 microdeletion, and 16p13.11microduplication, were reported in previous studies on miscarriage 21,37,42,43 .The possible underlying mechanism that results in early embryonic death may be a malformed fetal cardiovascular system resulting from 22q11.2 deletion and 7q11.23 deletion 6 .Whereas, whether 16p13.11microduplication contributes to miscarriage remains to be uncertain.In addition, microdeletions in 22q11.21,2q37.3 and 9p24.3p24.2 as well as CNVs in 11p15.5 were identified as likely to be associated with miscarriage 44 , the potential mechanism is that aberrant methylation or duplication of imprinted genes in 11p15.5 could cause miscarriage.Likewise, Nagirnaja et al. 45 identified CNV in 5p13.3,interrupting the PDZD2 and GOLPH3 genes, and showed that which was significant correlated with an increased risk of spontaneous abortion (SA).Recently, Sheng et al. 46 also identified two recurrent RSA-associated CNVs (duplications at 16q24.3 and 16p13.3)compared with the SA.
In our study, we also identified microdeletion in 2q37.3 and 22q11.2which may lead to miscarriage.In addition, eleven pCNVs, which were detected in CVSs at least twice, involved deletions at 1q21.1, 8p23.1, 2q37.3,5p15.33p14.3,7q32.3q36.3,17p12, 15q26.2q26.3,4p16.3p16.1, and Xp22.3q28, as well as duplications in 5p13 and 8q23.3q24.3.The association of miscarriage with other submicroscopic pCNVs identified in our cohort, is still unclear, and more large-scale studies are needed to confirm whether these submicroscopic pCNVs contribute to pregnancy loss.Recently, whole-exome sequencing has been applied to study RSA and a few embryonic lethal genes have been discovered 47 .We believe future large-scale cohort will identify more recurrent pCNVs and gene mutations concerning pregnancy loss.
Both SNP array and CNV-seq/QF-PCR were effective in identifying chromosomal aneuploidies, CNVs and polyploidies 48 .However, CNV-seq/QF-PCR and SNP array cannot detect all polyploidies, such as 69, XXX and tetraploidy, as well as balanced structural rearrangement.In addition, CNV-seq can detect mosaicism in at least 10% of CNVs, while microarray can detect mosaicism in at least 30% of CNVs and UPD.
Unlike embryonic chromosomal aneuploidies, the risk of pCNVs in aborted embryos is independent of maternal age 49 .Our data indicate that the incidence of other chromosomal abnormalities such as polyploidy, CNVs, and UPD was not directly correlated with maternal age (p > 0.05), which is partially consistent with the study reported by Larroya et al 50 .Based on this point, SNP array or CNV-seq/QF-PCR analysis on CVSs is strongly recommended, regardless of maternal age.
In the present study, the chromosomal structural abnormality rate (6.8%, 18/264) of CVSs samples in AMA was lower than that (12.6%, 113/896) of miscarriage samples in YMA, which is in line with a previous research 51 , because the frequency of embryotic chromosomal structural abnormalities did not seem to be correlated with AMA.A larger population study is needed to further clarify the noncorrelation.
UPD is a rare chromosomal abnormality.The major mechanism of UPD formation is trisomy rescue 52 .Specific regions on chromosomes 6, 7, 11, 14, 15, and 20 can result in genomic imprinting diseases.Chromosomewide UPD resulted from parthenogenesis, which is one of the genetic factors for early pregnancy loss 53 .The incidence (0.7%) of UPD in our study might have been underestimated since UPDs were not detected in the cases via CNV-seq/QF-PCR.Single-chromosome UPD is presumably suggestive of a monosomy rescue event.Segmental UPD in these cases would likely be due to a meiotic/mitotic error, in which a meiotic non-disjunction event was followed by a mitotic cross-over between the paternal and maternal homologs, with subsequent trisomy rescue 54 .Pregnancy loss could be due to UPD resulting in unmasking of an underlying lethal recessive disease gene(s) or imprinted genes.
The limitation of this study is that the number of cases is not large enough; the limited data is not fully representative of the CNV characteristics of early miscarriage.Thus, the correlations among the history of previous abortions/weeks of miscarriage, modes of conception, and embryonic chromosomal abnormalities have not yet been evaluated.For this, a larger population study and enrichment analysis of genetic functions within the examined CNVs will be necessary to identify the specific genes associated with miscarriage.
In summary, our study suggests both SNP array and CNV-seq/QF-PCR are reliable, robust, and high-resolution technologies for genetic diagnosis of miscarriage, especially in detecting CNVs.

Figure 4 .
Figure 4.The incidence of chromosomal abnormality for subgroups of different maternal age.

Figure 5 .
Figure 5.The number of chromosomal abnormality in subgroups of previous miscarriage.

Table 1 .
Demographic characteristics of 1160 cases with early missed abortion by molecular karyotyping analysis.

Table 2 .
Types and frequencies of chromosomal abnormalities detected in 1160 CVSs.Significant are in vaue [bold].CNV-seq copy number variation sequencing; QF-PCR quantitative fluorescent-polymerase chain reaction; SNP array single nucleotide polymorphism array; UPD uniparental disomy; VOUS variants of unknown significance.